Retroviruses pose a threat to human health by infecting somatic cells, but retroviruses have also been infecting our mammalian ancestors for millions of years, accumulating in the germ-line as ERVs that account for 10% of our genomic DNA. My laboratory studies ERVs from two perspectives: 1) as parasites that must be kept in check by the host to prevent widespread viral activation and 2) as symbionts that can be co-opted by the host for evolutionary advantage. Our long-term objective is to understand how the host has adapted recognition machinery to establish epigenetic silencing of ERVs, how ERVs sometimes evade these silencing mechanisms, and how this arms race between ERVs and the host have lead to co-option of viral regulatory sequences that may have contributed to the evolution of mammals. We hypothesize that the rapidly diversifying KRAB-ZFP family plays a critical role in the recognition and silencing of ERVs and nearby genes, and we are taking systematic genetic and biochemical approaches to explore this function. Kruppel associated box zinc finger proteins (KRAB-ZFPs) have emerged as candidates that recognize ERVs. KRAB-ZFPs are rapidly evolving transcriptional repressors that emerged in in a common ancestor of coelacanth, birds, and tetrapods. They make up the largest family of transcription factors in mammals (estimated to be several hundred in mice and humans). Each species has its own unique repertoire of KRAB-ZFPs, with a small number shared with closely related species and a larger fraction specific to each species. Despite their abundance, little is known about their physiological functions. KRAB-ZFPs consist of an N-terminal KRAB domain that binds the co-repressor KAP1 and a variable number of C-terminal C2H2 zinc finger domains that mediate sequence-specific DNA binding. KAP1 directly interacts with the KRAB domain, which recruits the histone methyltransferase (HMT) SETDB1 and heterochromatin protein 1 (HP1) to initiate heterochromatic silencing. Several lines of evidence point to a role for the KRAB-ZFP family in ERV silencing. First, the number of C2H2 zinc finger genes in mammals correlates with the number of ERVs. Second, the KRAB-ZFP protein ZFP809 was isolated based on its ability to bind to the primer binding site for proline tRNA (PBSPro) of murine leukemia virus (MuLV). Third, deletion of the KRAB-ZFP co-repressors Trim28 or Setdb1 leads to activation of many ERVs. Thus we have begun a systematic interrogation of KRAB-ZFP function as a potential adaptive repression system against ERVs. We focused on ZFP809 as a likely ERV-suppressing KRAB-ZFP since it was originally identified as part of a repression complex that recognizes infectious MuLV via direct binding to the 18 nt Primer Binding Site for Proline (PBSpro) sequence. We hypothesized that ZFP809 might function in vivo to repress other ERVs that utilized the PBSpro. Using ChIP-seq of epitope tagged ZFP809 in ESCs and embryonic carcinoma (EC) cells, we determined that ZFP809 bound to several sub-classes of ERV elements via the PBSpro. We generated Zfp809 knockout mice to determine whether ZFP809 was required for VL30pro silencing. We found that Zfp809 knockout tissues displayed high levels of VL30pro elements and that the targeted elements display an epigenetic shift from repressive epigenetic marks (H3K9me3 and CpG methylation) to active marks (H3K9Ac and CpG hypo-methylation). ZFP809-mediated repression extended to a handful of genes that contained adjacent VL30pro integrations. Furthermore, using a combination of conditional alleles and rescue experiments, we determined that ZFP809 activity was required in development to initiate silencing, but not in somatic cells to maintain silencing. These studies provided the first demonstration for the in vivo requirement of a KRAB-ZFP in the recognition and silencing of ERVs. As a follow-up to our studies on ZFP809, we have begun a systematic analysis of KRAB-ZFPs using a medium throughput ChIP-seq screen and functional genomics of KRAB-ZFP clusters and individual KRAB-ZFP genes. Our ChIP-seq data demonstrates that the majority of recently evolved KRAB-ZFP genes interact with and repress distinct and partially overlapping ERV targets. This is supported by a recent knockout mouse line lacking 17 KRAB-ZFPs (generated with CRISPR/Cas9 engineering) that displays an ERV reactivation phenotype. KRAB-ZFPs like ZFP809 initiate ERV silencing by establishing methylation of histone H3 lysine 9 via the recruitment of the histone methyltransferase SETDB1. Mice have three histone H3 variants (H3.1, H3.2 and H3.3), and we set out to explore whether one or more of these variants is critical for ERV silencing. Using ChIP-seq in primary mouse embryonic fibroblasts and induced pluripotent stem cells containing genetically tagged histone H3.3 genes, we found a strong enrichment of the variant histone H3.3 at ERVs co-occupied by KAP1, SETDB1, and H3K9me3, including VL30Pro elements recognized by ZFP809. Importantly, this enrichment was present only in pluripotent cells. Thus we explored the possibility that the deposition of histone H3.3 might be required for ERV silencing. To test this hypothesis, we used CRISPR/Cas9 to create a homozygous floxed Daxx gene, since DAXX had previously been shown to be responsible for H3.3 deposition at telomeres in ESCs. We found that acute loss of Daxx by Cre mediated recombination in ESCs caused a complete loss of histone H3.3 at ERVs, but had little effect on ERV reactivation in comparison to deletion of Setdb1, which leads to massive ERV reactivation. This data suggests that DAXX-dependent deposition of histone H3.3 is dispensable for ERV silencing. This finding is in conflict with a recent report that argued that some ERVs display reactivation in histone H3.3 KO ESCs, and that in particular, IAP elements become retro-transpositionally active. Our re-analysis of this dataset challenges this central conclusion. We found that there is no correlation between ERVs marked by histone H3.3 and those showing reactivation in H3.3 KOs; furthermore, we demonstrated that the reported IAP re-integrations are not due to retrotransposition, but were simply polymorphic IAP elements mixed into the genetic background of the ESCs used for the study. The authors of the study have acknowledged this error in their interpretation. Our data supports a model in which histone H3.3 is deposited into ERVs and other KRAB-ZFP target genes in pluripotent cells, but that this deposition is not required for ERV silencing, which has been supported further in the recent literature. Although our data shows that many KRAB-ZFPs repress ERVs, we also found that more ancient KRAB-ZFPs that emerged in a human/mouse common ancestor do not bind and repress ERVs. One of these KRAB-ZFPs, ZFP568 plays an important role in silencing a key developmental gene that may have played a critical role in the onset of viviparity in mammals. Using ChIP-seq and biochemical assays, we determined that ZFP568 is a direct repressor of a placental specific isoform of the Igf2 gene called Igf2-P0. Insulin-like growth factor 2 (Igf2) is the major fetal growth hormone in mammals. We demonstrated that loss of Zfp568, which causes gastrulation failure, or mutation of the ZFP568 binding site at the Igf2-P0 promoter causes inappropriate Igf2-P0 activation. We also showed that this lethality could be rescued by deletion of Igf2. These data highlight the exquisite selectivity by which members of the KRAB-ZFP family repress their targets and identifies an additional layer of transcriptional control of a key growth factor regulating fetal and placental development.